The central role of vascular endothelial growth factor (VEGF) in angiogenesis health and disease makes it attractive both as a therapeutic target for anti-angiogenic drugs in pathological conditions such as cancer and as a pro-angiogenic cytokine for the treatment of ischemic heart disease. VEGF modulates the complex process of angiogenesis and other various aspects of endothelial cell function through either of its two tyrosine kinase receptors, VEGFR1/Flt-1 or VEGFR2/Flk1/KDR via its target protein MK2. In the present proposal we will use Flk-1 , Flt-1 and MK2-/- (MAPKAPKinase2) knockout mice in an attempt to address an important clinical issue to identify potential downstream candidates of VEGF signaling through its receptors that trigger cardioprotective signal during ischemic preconditioning (IP). By Affymetrix gene chip analysis we demonstrated for the first time down regulation of genes (Pellino-1 or Peli-1, Epiregulin and NF?B) after ischemic insult to the Flk-1 mice compared to WT. Studies have identified candidates of the Pellino family as a novel upstream regulator in mediating activation of MAPKAP kinase pathway. PELLINO (Peli) and EPIREGULIN (Ereg) are the newly described molecules downstream of VEGF signaling which might play significant role in myocardial angiogenesis leading to the inhibition of ventricular remodeling. Ereg has angiogenic potential, which may contribute to de novo development of vessels by vasculogenesis or angiogenesis in ischemic/infarcted myocardium. Our long-term goal of this project is to understand the mechanism of VEGF signaling in the ischemic myocardium through newly described molecules Peli-1 and Ereg that we have explored in ischemic myocardium very recently. These are the molecules that may be involved in triggering PI3Kinase/MK2/NF?B pathway involved in angiogenesis. Specific Aim I will determine the involvement of Peli-1, 2 and 3 in IP mediated angiogenesis, Aim II will determine the involvement of MK2 and NF?B in Pellino mediated angiogenesis, Aim III will study the involvement of Ereg in VEGF/PI3-kinase/MK2/NF?B mediated angiogenesis, Aim IV will determine the role and involvement of NF?B in VEGF/Pellino/epiregulin/MK2 mediated myocardial angiogenesis. Genetically engineered NF?B knockout (KO) mice will be used to identify VEGF mediated signaling which may be through NF?B, one of the final target genes related to myocardial angiogenesis. This study will adapt multidisciplinary approach that will consist of various techniques such as modern molecular biology, imaging, gene targeting, siRNA technique and physiology. Collectively, the proposal will contribute to our understanding of the molecular mechanism of VEGF induced angiogenesis and in future may provide novel therapeutic treatment strategies against ischemic heart disease. PUBLIC HEALTH RELEVANCE: To understand VEGF signaling mechanism is a promising medical development in the treatment of ischemia that will likely improve the quality of life in patients who have severe ischemic heart disease.